Thermal cycler including a temperature gradient block

ABSTRACT

A method in which a temperature gradient is generated across a “gradient” block, and an apparatus comprising a block across which a temperature gradient can be generated. By setting up such a gradient, multiple reaction mixtures held in wells on the gradient block can be simultaneously run at temperatures which differ only slightly, thereby permitting an optimum temperature for the reaction to be quickly identified. In a preferred embodiment the gradient block is integrated into a thermal cycler used for nucleic acid amplification reactions.

FIELD OF THE INVENTION

[0001] The present invention relates to a temperature cycling apparatususeful for performing nucleic acid amplification, DNA sequencing and thelike which apparatus can include single or multiple heating and/orcooling blocks containing sample wells wherein a temperature gradientcan be generated across a given block.

BACKGROUND OF THE INVENTION

[0002] Systems which require multiple or cyclic chemical reactions toproduce a desired product often require careful temperature control toproduce optimal results. Such reactions include nucleic acidamplification reactions such as the polymerase chain reaction (PCR) andthe ligase chain reaction (LCR). For this reason, apparatus have beendeveloped which permit the accurate control of the temperature ofreaction vessels in which such amplification reactions are performed.

[0003] For example, there are a number of thermal “cyclers” used for DNAamplification and sequencing in the prior art in which one or moretemperature controlled elements or “blocks” hold the reaction mixture,and the temperature of a block is varied over time.

[0004] Another prior art system is represented by a temperature cyclerin which multiple temperature controlled blocks are kept at differentdesired temperatures and a robotic arm is utilized to move reactionmixtures from block to block.

[0005] All of these systems include features which allow the user toprogram temperatures or temperature profiles over time for a block onthe instrument so that various processes (e.g. denaturing, annealing andextension) can be efficiently accomplished once the optimum temperaturesfor these steps are determined. Importantly, however, the determinationof the optimum temperature for each of the various steps in any reactionsystem, and in particular for any nucleic amplification or incubationreaction involving an annealing step, is not a simple task.

[0006] PCR is a technique involving multiple cycles that results in thegeometric amplification of certain polynucleotide sequence each time acycle is completed. The technique of PCR is well known to the person ofaverage skill in the art of molecular biology. The technique of PCR isdescribed in many books, including, PCR: A Practical Approach, M. J.McPherson, et al., IRL Press (1991), PCR Protocols: A Guide to Methodsand Applications, by Innis, et al., Academic Press (1990), and PCRTechnology: Principals and Applications for DNA Amplification, H. A.Erlich, Stockton Press (1989). PCR is also described in many U.S. Pat.Nos., including U.S. Pat. Nos., 4,683,195; 4,683,202; 4,800,159;4,965,188; 4,889,818; 5,075,216; 5,079,352; 5,104,792; 5,023,171;5,091,310; and 5,066,584, which are hereby incorporated by reference.

[0007] The PCR technique typically involves the step of denaturing apolynucleotide, followed by the step of annealing at least a pair ofprimer oligonucleotides to the denatured polynucleotide, i.e.,hybridizing the primer to the denatured polynucleotide template. Afterthe annealing step, an enzyme with polymerase activity catalyzessynthesis of a new polynucleotide strand that incorporates the primeroligonucleotide and uses the original denatured polynucleotide as asynthesis template. This series of steps (denaturation, primerannealing, and primer extension) constitutes a PCR cycle. As cycles arerepeated, the amount of newly synthesized polynucleotide increasesgeometrically because the newly synthesized polynucleotides from anearlier cycle can serve as templates for synthesis in subsequent cycles.Primer oligonucleotides are typically selected in pairs that can annealto opposite strands of a given double-stranded polynucleotide sequenceso that the region between the two annealing sites is amplified.

[0008] The temperature of the reaction mixture must be varied during aPCR cycle, and consequently varied many times during a multicycle PCRexperiment. For example, denaturation of DNA typically takes place ataround 90-95° C., annealing a primer to the denatured DNA is typicallyperformed at around 40-60° C., and the step of extending the annealedprimers with a polymerase is typically performed at around 70-75° C.Each of these steps has an optimal temperature for obtaining the desiredresult. Many experiments are required to determine the optimaltemperature for each step.

[0009] For example, while the temperature at which DNA denatures isgenerally between 90-95° C., slight variations in the particulartemperature necessary are observed depending on the length of the DNAand the percentage of each of the four deoxynucleotides present(guanine-cytosine pairs and adenine-thymine pairs). Insufficient heatingduring the denaturation step is a common reason for a PCR reaction tofail. However, overheating during the denaturation step can result inexcessive denaturation of the polymerase.

[0010] Achieving the optimal temperature for the PCR annealing step iseven more critical. An annealing temperature which is too low willresult in non-specific DNA fragments being amplified. At too high of anannealing temperature, the primers will anneal less efficientlyresulting in decreased yield of the desired product and possibly reducedpurity. In the annealing step, the optimal temperature will depend onmany factors including the length of the primer and the percentage ofeach of the four deoxynucleotides present (guanine-cytosine pairs andadenine-thymine pairs). For a typical 20-base oligonucleotide primercomprised of roughly 50% guanine-cytosine, a temperature of 55° C. is agood estimate for the lower end of the temperature range. However, asone increases the primer length in order to attain greater primerspecificity, differing annealing temperatures may be required. Thus, thenumber of subtle influences on the optimal annealing temperature makesdifficult the task of quickly identifying the optimum for a givensystem,

[0011] Achieving the optimal temperature for the extension reaction isalso important for obtaining the desired PCR result. Temperature mayaffect both the rate and the accuracy of the extension reaction. If therate of the polymerase reaction is too low, then the newly synthesizedpolynucleotide may not contain a site for primer annealing.Additionally, the denatured polynucleotide sequence for amplificationmay contain one or more regions of secondary structure that may form ordisappear according to the temperature selected. Furthermore, severaldifferent enzymes with polymerase activity may be used for PCR. Eachenzyme will have its own optimum temperature for activity, stability andaccuracy.

[0012] Determination of the optimal denaturing, annealing, and extensiontemperatures for a particular PCR is complicated by the fact that theoptimum will be different for each of the reactions. Thus, in order todetermine the three optimal temperature ranges, multiple separateexperiments must be run where two temperature variables are heldconstant while a third temperature variable is changed. As a result,determination of the optimal temperature for running a PCR system can bea time consuming task.

[0013] It is therefore an object of the present invention to provide anefficient means by which optimal reaction temperatures can be moreefficiently identified for PCR and other reactions.

SUMMARY OF THE INVENTION

[0014] To achieve this object, the invention is a method in which atemperature gradient is generated across a “gradient” block. Theinvention also includes an apparatus comprising a block across which atemperature gradient can be generated. By setting up such a gradient,multiple reaction mixtures can be simultaneously run at temperatureswhich differ only slightly, thereby permitting an optimum temperaturefor a given reaction to be quickly identified. In the most preferredembodiment of the invention the gradient block is integrated into athermal cycler. By doing so, it is possible to run a series of desiredreactions using the thermal cycler immediately upon identification ofthe optimum reaction temperature.

[0015] In a first embodiment, the invention is a method forsimultaneously reacting a plurality of reaction mixtures in an apparatusincluding a temperature gradient block comprising the steps of:

[0016] placing reaction mixtures in a plurality of reaction wells in thegradient block, the gradient block having a top portion, first andsecond oppposing portions, and a bottom portion, the plurality ofreaction mixture wells being formed in the block between the opposingportions, and

[0017] generating a temperature gradient across said gradient block andbetween the opposing portions.

[0018] In this embodiment, the step of generating a temperature gradientmay comprise the steps of heating the first opposing portion of thegradient block, and cooling the second opposing portion of the gradientblock. The method may also include the step of controlling thetemperature gradient using a controlling means. By using a controllingmeans, the method may further include the steps of collecting andstoring temperature set point and actual temperature data from thewells, and transmitting that information to a microprocessor.

[0019] In another form of the method of the invention, where theapparatus further comprises at least one additional heat conductingblock having a top portion, first and second opposing portions, and abottom portion, and a plurality of reaction mixture wells formed in theadditional block between the opposing portions, the method may furthercomprise the step of moving the reaction mixtures between the gradientblock and one or more of the additional block or blocks.

[0020] In another form, the method employs an apparatus comprising atleast one heat conducting block, the block having a plurality of samplewells spaced between first and second opposing portions and in an uppersurface thereof, and the method comprises

[0021] placing reaction mixtures in the wells, and

[0022] generating a temperature gradient across the block and betweenthe opposing portions by heating the first opposing portion and coolingthe second opposing portion.

[0023] The invention also includes an apparatus comprising:

[0024] a reaction mixture holder, the reaction mixture holder comprisinga heat conducting block having a top portion, first and second opposingportions, and a bottom portion, a plurality of reaction mixture wellsformed in the top portion, and between the first and second opposingportions,

[0025] a block heater positioned adjacent to the first opposing portion,and

[0026] a block cooler positioned adjacent to the second opposingportion.

[0027] In another form, the apparatus of the invention comprises holdingmeans for holding a reaction mixture, the holding means including a heatconducting block having a top portion, first and second opposingportions, and a bottom portion, and a plurality of reaction mixturewells formed in the top portion and between the first and secondopposing portions; and means for generating a temperature gradientacross the heat conducting block and between the first and secondopposing portions.

[0028] In yet another form, the invention includes an apparatus forperforming molecular biological reactions comprising at least onetemperature controlled block, the block having a plurality of reactionmixture wells spaced between first and second opposing portions and inan upper portion thereof, and a block heater positioned adjacent to thefirst opposing portion and capable of generating a temperature gradientbetween the first and second opposing portions.

[0029] In a preferred embodiment, the heat conducting block or“gradient” block is made substantially of, or comprises, brass.

[0030] The apparatus of the invention can include additional elements.Thus, in an especially preferred embodiment, the apparatus includes morethan one heat conducting block along with the gradient block. Theapparatus may also include a controller for controlling the temperaturegradient across the gradient block, and in a multi-block apparatus, thecontroller may also control the temperature of blocks which are heatedor cooled to a uniform temperature. Preferably, the controller willinclude a microprocessor for collecting and storing temperature setpoint and actual temperature data, and multiple temperature sensors forcollecting the actual temperature data from the wells and fortransmitting the information to the microprocessor.

[0031] In another embodiment, the plurality of wells in the gradientblock are formed in parallel, aligned rows. Further, where more than oneblock is included, the apparatus may include a robot arm for movingsamples between blocks in a programmably controllable manner.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The invention will be better understood by reference to theappended figures of which:

[0033]FIG. 1 is a perspective view of a thermal cycler incorporating thethermal gradient block of the invention;

[0034]FIG. 2 is a perspective, exploded view, of the thermal gradientblock, surrounding heaters and cooler according to the invention; and

[0035]FIG. 3 is a block diagram depicting the elements of a thermalcycler in which the thermal gradient apparatus and method of theinvention may be used.

DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention relates to a device and method for creatinga thermal gradient across a block, such as a block in known thermalcyclers for PCR reactions, which enables one to simultaneously conduct aseries of experiments at very close to the same temperatures. As usedherein, the term “block” refers to a structure, usually metal, which canbe temperature controlled and in which wells have been arranged toaccept vessels containing reaction mixtures or “samples.” The phrase“gradient block” as used herein is intended to describe such a block,except that a gradient block is a block across which a temperaturegradient can be established. Examples of the specific manner in whichsuch a temperature gradient can be established are discussed herein,though those skilled in the art will understand that once the advantageof having a gradient block is known, many other variations of theapparatus shown herein can be easily identified.

[0037] One particular area of utility for the present invention is inmultiple block thermal cyclers. By incorporating the gradient block ofthe invention into a multiple block thermal cycler, it is possible tosimultaneously conduct a series of reactions where the temperature atwhich the reactions are proceeding is varied across the gradient block.This permits the rapid determination of the optimal temperature for thatparticular reaction.

[0038]FIG. 1 depicts the prior art Stratagene device in which a thermalgradient block 2 according to the invention has been incorporated.

[0039] Various components of the cycler depicted in greater detail inFIGS. 2 and 3 can be seen in FIG. 1, i.e. display 15, keypad 16,additional blocks 17, 18 and 19 and robot arm 20 (shown in cut-awayview).

[0040] It will be understood that a microprocessor can is beincorporated into the control electronics of the apparatus, as is wellknown. The microprocessor can be used to control the range of thetemperature gradient and also to program the movement of samples intoand out of the thermal gradient block. The microprocessor executescommands written in software that collect user input via the keyboard,compare the input to actual temperatures, and turn off or on the heatingor cooling units as appropriate. The electronics also includes a timer,readable by the microprocessor. This allows the microprocessor tocompare the elapsed time that the reaction mixture has been in a givenblock and compare it to a desired time input by the user.

[0041] The microprocessor will also control the robot arm, which arm isoperated using two stepper motors. One motor raises and lowers the arm.The other rotates the arm from block to block.

[0042] Thus, those skilled in the art can readily understand how thethermal gradient block of the invention can be incorporated into knownthermal cyclers.

[0043] Of course, the thermal gradient block of the invention need notnecessarily be incorporated into a known cycler to be advantageouslyused. For example, a stand alone unit incorporating the thermal gradientblock could be used in conjunction with known cyclers so that optimumreaction temperatures could be identified and then used in thosecyclers.

[0044]FIG. 2 provides an exploded view of the components of the gradientblock assembly. Thus, in FIG. 2, the gradient block apparatus isgenerally designated by reference number 1. The apparatus comprises aheat conducting block 2 which incorporates a number of wells 3 forholding reaction mixtures or the vessels in which the mixtures can beheld. In a portion of block 2, heater 5 fits into opening 4. Heater 5 isa commonly available cylindrically shaped cartridge type resistiveheater (RAMA brand, San Jacinto, Calif.).

[0045] Depending on the temperature range desired, the opposing portionof block 2 may simultaneously be cooled using a heat sink made up of aribbed aluminum block 7 and a fan 9. Naturally, whether the heat sink isoperated or not, a temperature gradient will be created between theopposing portions of the block. However, where the temperature gradientis to be made larger, the heat sink can be operated. To enhance theability to create and maintain a gradient, block 2 is preferablycomposed of a material with a relatively low coefficient of thermalconductivity to reduce the amount of heat flux necessary to create thetemperature gradient across the block. Brass is preferred.

[0046] Where a multiblock system is used (FIG. 1) blocks other than thegradient block will be constructed of a material with a relatively highcoefficient of thermal conductivity. By doing so, the blocks can beheated or cooled to a uniform temperature but will not be thermallyconductive enough to require excess heating or cooling to maintain atemperature. Aluminum is known for such uses in the prior art.

[0047] Depending on the size of the gradient block and the heating andcooling capacities of the heater and heat sink, temperature gradients inexcess of 1 to 14° C. across block 2 can be achieved. Holes 6 may bedrilled in block 2 to limit thermal conductivity, such that parallelaligned rows of wells in the block tend to be at one temperature. Theuse of holes also permits the temperature profile across the gradientblock, and from one row of wells to the next, to be linear.

[0048] Heaters and coolers known in the art may be used. For example,Peltier thermoelectric devices may be used, though other passive oractive heaters would also be useful (e.g. chilled or heated liquids orgases).

[0049] As shown in both FIGS. 1 and 2, gradient block 2 preferably haseight rows of sample wells 3 equally spaced across the block. Each rowcan contain five sample wells. 0.5 ml tubes can be used. The particularnumber and design of the sample wells can be varied to modify capacityif desired. If a temperature gradient of 8° C. is formed between the hotand cold portions of the block, each row of sample wells will differ intemperature by approximately 1° C.

[0050] Returning to FIG. 2, additional heaters 8 and 10 may also beemployed so that the system can be operated in the same manner as blocksknown in the art, i.e. with uniform heating across the entire length andwidth of block 2. Heaters 8 and 10 are preferrably thin foil type (MINCObrand Minneapolis, Minn.). Heaters 8 and 10 can also be in conjunctionwith heater 5 to bring block 2 to at least the cool portion temperatureas quickly as possible when the system is started or the temperaturerange over which block 2 is to be operated is raised.

[0051] Wire connectors 11, 12 and 13 connect the heaters to a powersource. Apparatus 1 may also include a thermostat 14 which can be usedas a high temperature cut-off, which is a desirable safety feature.

[0052] The block diagram of FIG. 3 depicts a gradient block (labelled“second block”) of the type shown in FIG. 2 as block 2 integrated into athermal cycler having multiple heating and cooling blocks. The labels inFIG. 3 are self-explanatory, and the apparatus described by FIG. 2differs from a known thermal cycler only with respect to thesubstitution of the gradient block for a non-gradient block. For PCR,the first, second and third blocks in FIG. 3 may be programmed to bemaintained at a temperature range of between about 25 to 99° C., and areused for denaturing, annealing and extension respectively. The fourthblock is generally maintained at between 4 and 25° C. and is used forsample storage after the PCR reaction has completed. The second block,made of brass, will be used for the annealing step.

[0053] As can be seen in FIG. 3, more than one thermocouple can be usedalong the length of the gradient block so that temperatures along theblock can be carefully monitored and used to feed information back tothe control electronics and display.

[0054] The following examples are offered for the purpose ofillustrating, not limiting, the subject invention.

EXAMPLE 1 Use of the Gradient Thermal Cycler for the Polymerease ChainReaction

[0055] High temperature primer extension testing of the thermal gradientsystem of the invention was carried out using two model primer/templatesystems. These two systems exhibit significantly variable extensionproduct yields depending upon the annealing temperature used during theextension process. Primer/template set #1 amplifies a 105 bp region ofthe human Gaucher gene, while set #2 amplifies a 540 bp region of thehuman fucosidase gene. The thermal gradient system of the inventioncontains a gradient block that allowed primer extension using an optimalannealing temperature range of 42 to 56° C.

[0056] Methods and Materials

[0057] Primer extension reactions were carried out using the gradientblock of the invention. Primer/template test set #1 consisted of agenomic human DNA template and two 22mer oligomers yielding a 105 bpextension product. The sequence of primer A was 5′CCTGAGGGCTCCCAGAGAGTGG 3′9 (SEQ ID NO:1). The sequence of primer B was5′ GGTTTAGCACGACCACAACAGC 3′ (SEQ ID NO:2). Primer/template test set #2consisted of a genomic human DNA template and two oligomers of 20 and 30bases respectively yielding a 540 bp extension product. The sequence ofprimer A was 5′ AGTCAGGTATCTTTGACAGT 3′ (SEQ ID NO:3). The sequence ofprimer B was 5′ AAGCTTCAGGAAAACAGTGAGCAGCGCCTC 3′ (SEQ ID NO:4).

[0058] The primer extension reaction mixture consisted of 1×Taq DNApolymerase buffer (10 mM tris-HCl pH 8.8, 50 mM KCl, 1.5 mM MgCl2, .001%(w/v) gelatin), 250 uM each dNTP, 250 ng each primer and template and2.5 units Taq DNA polymerase in a 100 μl reaction volume. The reactionmixture was overlayed with 50 μl of nuclease free sterile mineral oil.

[0059] The temperature cycling parameters used were as follows: 1 min94° C. 1 min 42-56° C. (gradient block) | | 1 min 72° C. | 1 min 94° C.| 30 cycles 1 min 42-56° C. (gradient block) | | 8 min 72° C. Storage 4° C.

[0060] Eight reaction mixes were tested per primer/template set—one pergradient temperature block slot. Annealing temperatures used were 42,44, 46, 48, 50, 52, 54 and 56° C. (two degree C increments across thegradient block). Reactions were carried out in 500 μl eppendorf tubes.

[0061] Results

[0062] Both primer/template sets 1 and 2 yielded obviously varyingresults depending upon the annealing temperature used in the gradienttemperature block. Primer extension products from set #1 varied from thedesired single DNA band of size 105 bp (derived from the extensionreaction using a 56° C. annealing temperature) to a reaction mixyielding multiply sized extraneous DNA extension products (ofapproximate size 180, 280 and 800 bp) from a reaction using a 48° C.annealing temperature. Primer extension products from set #2 varied fromthe desired single DNA band of size 540 bp (derived from the extensionreaction using a 42° C. annealing temperature) to a reaction mixyielding an extraneous DNA extension product of approximately 2000 bpfrom a reaction using a 56° C. annealing temperature.

EXAMPLE 2 Use of the Gradient Thermal Cycler for the Ligase ChainReaction

[0063] Ligase chain reaction (LCR) is a recently described DNAamplification technique that can be used to detect trace levels of knownnucleic acid sequences. LCR involves a cyclic two step reaction which isperformed in a DNA thermal cycler machine. The first step is a hightemperature melting step in which double stranded DNA unwinds to becomesingle stranded. The second step is a cooling step in which two sets ofadjacent, complementary oligonucleotides anneal to the single strandedtarget DNA molecules and are ligated together by a DNA ligase enzyme.The products of ligation from one cycle serve as templates for theligation reaction of the next cycle. Thus, LCR results in theexponential amplification of ligation products.

[0064] Materials and Methods

[0065] The materials used in this experiment were obtained fromStratagene, La Jolla, Calif. The optimal temperature for the second stepof the LCR cycle, in which the oligonucleotides are annealed to the DNAtarget molecules, was determined empirically by the use of the gradientthermal cycler of the invention. Two sets of reactions were set up, onewith a wild type template to which the oligonucleotides werecomplementary, and one with a mutant template that differed from thewild type template DNA sequence by one base transition. The DNAtemplates used in this experiment were plasmid constructs containing thepBluescriptII vector and the lac I gene. The wild-type templatecontained a normal lac I sequence, and the mutant template contained a Cto T transition mutation at site 191 within the insert. The fouroligonucleotide probes consisted of two pairs of two oligonucleotideseach. The first set, A and B, were adjacent to each other andcomplementary to one strand of the target DNA. The second set, C and D,were complementary to the first set, and therefore occupied adjacentsites on the second strand of the target DNA. The oligonucleotide probesequences (5′ to 3′) were as follows: A: TTGTGCCACG CGGTTGGGAA TGTA (SEQID NO:5) B: AGCAACGACT GTTTGCCCGC CAGTTC (SEQ ID NO:6) C: TACATTCCCAACCGCGTGGC ACAAC (SEQ ID NO:7) D: AACTGGCGGG CAAACAGTCG TTGT (SEQ IDNO:8)

[0066] Oligonucleotide probes A and D were 5′-phosphorylated duringsynthesis.

[0067] The sequence of the wild type lac I insert, starting at site 161of the insert, was as follows: 5′ CTGAATTACA TTCCCAACCG CGTGGCACAACAACTGGCGG GCAAACAGTC GTTGCTGATT 3′ (SEQ ID NO:9)

[0068] The mutant sequence differed from the wild type by a C to Ttransition at site 191.

[0069] The LCR experiment was performed as follows: The followingingredients were combined in a sterile 500-μl of 10×Z Pfu LCR buffer, 15μl of sterile dH₂O, 1 μl (10 ng of each) of oligonucleotide mixture, 1μl (100 pg) of either the wild-type or mutant plasmid templates or notemplate, and 1 μl (4U) of Pfu DNA ligase enzyme. A 25 -μl overlay ofsterile mineral oil was added to the tube. This procedure was repeatedso that there were a total of 5 tubes each of either the wild typetemplate reaction mixture or the mutant template reaction mixture. Thetubes were placed in the gradient thermal cycler of the invention inpositions 1, 3, 5, 7 and 8, so that at each isothermal column in themachine, there would be a wild type and a mutant template reaction. Themachine was programmed to cycle between a high temperature of 92° C. andthe gradient block, which was varied in temperature between 56° C. and70° C. The machine was programmed to move to the high temperature blockfor 4 minutes, then the gradient block for 3 minutes, then to movebetween the high temperature block and the gradient block 25 times,stopping for 1 minute at each block. The ligation chain reactionproducts were visualized by electrophoresis on a 6% polyacrylamide getbuffered with TBE, followed by staining with ethidium bromide andphotography under UV light.

[0070] Results

[0071] The wild type template reaction produced the most intensepositive signal in position 8, which corresponds to the coldest (56° C.)section of the gradient block. The use of the gradient thermal cycler ofthe invention allowed the empirical determination of the best annealingtemperature for this reaction in one experiment.

[0072] There are many modifications and variations of the thermalgradient block which can advantageously be incorporated into it orrelated structures. Further, multiple thermal gradient blocks could beemployed as more than one block of a multi-block thermal cycler wheresamples are automatically moved between the various blocks, therebyallowing for multiple reactions to be operated at multiple temperatures.

[0073] The invention has been described in detail with respect to itsuse with PCR. However, in addition to being exceptionally useful for thedetermination of the optimal temperature for individual steps in a PCRprocedure, the invention is also useful for determining the optimaltemperature for numerous other chemical reactions. These other chemicalreactions include any non-PCR nucleic acid amplification that employs anannealing step analogous to a PCR annealing step, such as ligase chainreaction (LCR) and DNA cycle sequencing. Other types of reactions forwhich the invention will be useful include DNA sequencing, cDNAsynthesis using a cycling reaction, coupled amplification sequencing(CAS), rapid amplification of cDNA ends (RACE) and any other incubationreaction in which incubations must be accomplished at multipletemperatures.

1 9 22 base pairs nucleic acid single linear DNA (genomic) NO NO 1CCTGAGGGCT CCCAGAGAGT GG 22 22 base pairs nucleic acid single linear DNA(genomic) NO NO 2 GGTTTAGCAC GACCACAACA GC 22 20 base pairs nucleic acidsingle linear DNA (genomic) NO NO 3 AGTCAGGTAT CTTTGACAGT 20 30 basepairs nucleic acid single linear DNA (genomic) NO NO 4 AAGCTTCAGGAAAACAGTGA GCAGCGCCTC 30 24 base pairs nucleic acid single linear DNA(genomic) NO NO 5 TTGTGCCACG CGGTTGGGAA TGTA 24 26 base pairs nucleicacid single linear DNA (genomic) NO NO 6 AGCAACGACT GTTTGCCCGC CAGTTC 2625 base pairs nucleic acid single linear DNA (genomic) NO NO 7TACATTCCCA ACCGCGTGGC ACAAC 25 24 base pairs nucleic acid single linearDNA (genomic) NO NO 8 AACTGGCGGG CAAACAGTCG TTGT 24 60 base pairsnucleic acid single linear DNA (genomic) NO NO 9 CTGAATTACA TTCCCAACCGCGTGGCACAA CAACTGGCGG GCAAACAGTC GTTGCTGATT 60

What is claimed is:
 1. A method for simultaneously reacting a pluralityof reaction mixtures in an apparatus including a temperature gradientblock comprising the steps of: placing reaction mixtures in a pluralityof reaction wells in said gradient block, said gradient block having atop portion, first and second oppposing portions, and a bottom portion,said plurality of reaction mixture wells being formed in said blockbetween said opposing portions, and generating a temperature gradientacross said gradient block and between said opposing portions.
 2. Amethod according to claim 1 Wherein said step of generating atemperature gradient comprises the steps of heating said first opposingportion of said gradient block, and cooling said second opposing portionof said gradient block.
 3. A method according to claim 1 including thefurther step of controlling said temperature gradient using controllingmeans.
 4. A method according to claim 3,wherein said controlling stepcomprises the steps of collecting and storing temperature set point andactual temperature data from said wells, and transmitting saidinformation to a microprocessor.
 5. A method according to claim 1wherein said apparatus further comprises at least one additional heatconducting block having a top portion, first and second opposingportions, and a bottom portion, and a plurality of reaction mixturewells formed in said additional block between said opposing portions,the method further comprising the step of moving said reaction mixturesbetween said gradient block and said additional block or blocks.
 6. Amethod for automated temperature cycling of a reaction mixture using athermal cycling apparatus comprising at least one heat conducting block,said block having a plurality of sample wells spaced between first andsecond opposing portions and in an upper surface thereof, the methodcomprising placing reaction mixtures in said wells, and generating atemperature gradient across said block and between said opposingportions by heating said first opposing portion and cooling said secondopposing portion.
 7. An apparatus for generating a temperature gradientacross a heat conducting block comprising: a reaction mixture holder,said reaction mixture holder comprising a heat conducting block having atop portion, first and second opposing portions, and a bottom portion, aplurality of reaction mixture wells formed in said top portion, andbetween said first and second opposing portions, a block heaterpositioned adjacent to said first opposing portion, and a block coolerpositioned adjacent to said second opposing portion.
 8. An apparatus forgenerating a temperature gradient across a heat conducting blockaccording to claim 7 wherein said apparatus further comprises controllermeans for controlling said block heater and block cooler.
 9. Anapparatus for generating a temperature gradient across a heat conductingblock according to claim 7 wherein said heat conducting block comprisesbrass.
 10. An apparatus for generating a temperature gradient across aheat conducting block according to claim 8 wherein said controller meanscomprises a microprocessor for collecting and storing temperature setpoint and actual temperature data.
 11. An apparatus for generating atemperature gradient across a heat conducting block according to claim 7wherein said plurality of wells in said heat conducting block are spacedacross said top portion.
 12. An apparatus for generating a temperaturegradient across a heat conducting block according to claim 11 whereinsaid plurality of wells in said heat conducting block are spaced acrosssaid top portion in parallel, aligned rows.
 13. An apparatus forgenerating a temperature gradient across a heat conducting blockaccording to claim 7 wherein said holder further comprises a heaterpositioned adjacent to said bottom portion.
 14. An apparatus forgenerating a temperature gradient across a heat conducting blockcomprising holding means for holding a reaction mixture, said holdingmeans comprising: (i) a heat conducting block having a top portion,first and second opposing portions, and a bottom portion, and aplurality of reaction mixture wells formed in said top portion andbetween said first and second opposing portions; and (ii) means forgenerating a temperature gradient across said heat conducting block andbetween said first and second opposing portions.
 15. An apparatus forgenerating a temperature gradient across a heat conducting blockaccording to claim 14 wherein said means for generating a temperaturegradient comprises means for heating said first opposing portion of saidblock, and means for cooling said second opposing portion of said block.16. An apparatus for generating a temperature gradient across a heatconducting block according to claim 14 wherein said apparatus furthercomprises at least one additional holding means for holding a reactionmixture, said additional holding means comprising: (i) at least oneadditional heat conducting block including a plurality of reactionmixture wells; and (ii) means for heating said additional heatconducting block.
 17. An apparatus for generating a temperature gradientacross a heat conducting block according to claim 14 wherein said heatconducting block comprises brass.
 18. An apparatus for generating atemperature gradient across a heat conducting block according to claim14 wherein said plurality of wells in said heat conducting block arespaced across said block in parallel, aligned rows.
 19. An apparatus forgenerating a temperature gradient across a heat conducting blockaccording to claim 14 wherein said apparatus further comprisescontroller means for generating said temperature gradient.
 20. Anapparatus for generating a temperature gradient across a heat conductingblock according to claim 19 wherein said controller means comprises amicroprocessor for collecting and storing temperature is set point andactual temperature data.
 21. An apparatus for generating a temperaturegradient across a heat conducting block according to claim 14 whereinsaid holder means further comprises a heating means for heating saidbottom portion.
 22. An apparatus for generating a temperature gradientacross a heat conducting block according to claim 16 wherein saidapparatus further comprises robot arm means, controlled by robot armcontrol means, for moving said reaction mixture between said holdingmeans.
 23. An automated apparatus for performing molecular biologicalreactions comprising at least one temperature controlled block, saidblock having a plurality of reaction mixture wells spaced between firstand second opposing portions and in an upper portion thereof, and ablock heater positioned adjacent to said first opposing portion andcapable of generating a temperature gradient between said first andsecond opposing portions.